Fig 1.
Overview of methionine synthesis.
(A) The standard pathway with 5-methyl-THF. (B) The reaction catalyzed by the core methionine synthases MesA and MesB. (C) The structure of methylcobalamin. Cobalamin has 5,6-dimethylbenzimidazole as the lower ligand, but many organisms use cobamides with other lower ligands. (D) The structure of 5-methyl-THF. Although THF is shown with a single glutamyl residue (at right), in the cell, THF is usually polyglutamylated.
Fig 2.
The proposed pathway for methionine synthesis in Dehalococcoides mccartyi.
Steps that are absent from D. mccartyi are shown with a red x.
Fig 3.
Comparative genomics links MesB to the Wood-Ljungdahl pathway.
(A) A phylogenetic tree of MesB and related proteins. The MesB family is highlighted in green and a subfamily that lacks Zn-coordinating residues is highlighted in red. On the right, filled symbols indicate the presence in that genome of other methionine synthases or of the Wood-Ljungdahl pathway (acsBCD, also known as cdhCED). If the genome contains more than one mesB gene, we show the number. The tree and the genome properties were rendered with iTOL v5 (https://itol.embl.de/). (B) Conserved clustering of mesB with genes from the Wood-Ljungdahl pathway. Gene drawings were modified from MicrobesOnline [21].
Fig 4.
Functional residues of MetE and of core methionine synthases.
We show sequence logos [33] for the zinc-coordinating and substrate-binding residues of each family of methionine synthases. The height of each position shows its conservation within the family, as measured by information content or bits. In MetE from E. coli, the zinc-coordinating residues are H641, C643, E665, and C726, and the substrate-binding residues are S433, E484, and D599.
Fig 5.
Sphingomonas koreensis can grow in minimal media by using MesD and not MetF.
A pool of transposon mutants was grown in a defined minimal media with a single carbon source and without added vitamins. Some cultures were supplemented with 250 μM L-methionine. Each cell in the heatmap shows a gene fitness value from a different experiment; each condition has two replicates. A gene fitness value is the log2 change in the relative abundance of mutants in that gene during that experiment (from inoculation at OD600 = 0.02 until saturation).
Fig 6.
Complementation assays show that MesD requires MesX and oxygen for activity, but not MetF.
We cloned MesD, MesX, or MesD and MesX together into strains of E. coli from the Keio collection [40] and measured growth in minimal glucose M9 medium. A plasmid bearing red fluorescent protein (RFP) was used as a control.
Table 1.
Oligonucleotide sequences.